A Human Mutation in Gabrg2 Associated with Generalized Epilepsy Alters the MembraneDynamics of GABAA Receptors

Walid Bouthour1,2,3, Felix Leroy1,2,3, Charline Emmanuelli1,2,3, Michele Carnaud1,2,3, Maxime Dahan4, Jean Christophe Poncer1,2,3 and

Sabine Levi1,2,3

1Institut National de la Sante et de la Recherche Medicale, Unite Mixte de Recherche en Sante 839, 75005 Paris, France, 2Universite

Pierre et Marie Curie, 75005 Paris, France, 3Institut du Fer a Moulin, 75005 Paris, France and 4Laboratoire Kastler Brossel, Centre

National de la Recherche Scientifique Unite Mixte de Recherche 8552, Physics Department and Institute of Biology, Ecole Normale

Superieure, 75005 Paris, France.

Bouthour and Leroy contributed equally to this work

Address correspondence to Sabine Levi, Institut National de la Sante et de la Recherche Medicale, Unite Mixte de Recherche en Sante 839, 17 rue du

Fer a Moulin, 75005 Paris, France. Email: sabine.levi@inserm.fr.

Neuronal activity modulates the membrane diffusion of post-synaptic g-aminobutyric acid (GABA)A receptors (GABAARs),thereby regulating the efficacy of GABAergic synapses. TheK289M mutation in GABAARs subunit g2 has been associated withthe generalized epilepsy with febrile seizures plus (GEFS1)syndrome. This mutation accelerates receptor deactivation andtherefore reduces inhibitory synaptic transmission. Yet, it is notclear why this mutation specifically promotes febrile seizures. Weshow that upon raising temperature both the number of GABAARsclusters and the frequency of miniature inhibitory postsynapticcurrents decreased in neurons expressing the K289M mutant butnot wild-type (WT) recombinant g2. Single-particle trackingexperiments revealed that raising temperature increases themembrane diffusion of synaptic GABAARs containing the K289Mmutant but not WT recombinant g2. This effect was mediated byenhanced neuronal activity as it was blocked by glutamate receptorantagonists and was mimicked by the convulsant 4-aminopyridine.Our data suggest the K289M mutation in g2 confers GABAARs withenhanced sensitivity of their membrane diffusion to neuronalactivity. Enhanced activity during hyperthermia may then trigger theescape of receptors from synapses and thereby further reduce theefficacy of GABAergic inhibition. Alteration of the membranediffusion of neurotransmitter receptors therefore represents a newmechanism in human epilepsy.

Keywords: epilepsy, GABA, GABAA receptor, hippocampus, quantum dots,single particle tracking

Introduction

c-aminobutyric acid (GABA) acting on GABAA receptors

(GABAARs) mediates fast inhibitory synaptic transmission in

the brain. Most GABAARs at cortical synapses are heteropen-

tamers of a1-3, b2/3, and c2 subunits (Luscher and Keller

2004). c2 subunit is required for benzodiazepine binding

(Mohler et al. 2002). It also affects the kinetics and

conductance of GABAAR channels (Gunther et al. 1995) and

is required for postsynaptic clustering (Essrich et al. 1998) and

synaptic maintenance (Schweizer et al. 2003). Mutations in the

Gabrg2 gene, encoding the c2 subunit, have been associated

with generalized epilepsy syndromes including febrile seizures

(FS) and generalized epilepsy with febrile seizures plus (GEFS+)(Macdonald et al. 2010). In particular, the missense mutation

K289M shows autosomal dominant inheritance and affects

a conserved residue in the short extracellular loop between

transmembrane domains II and III. Although this mutation was

initially suggested to reduce both membrane expression of c2(Kang et al. 2006) and the amplitude of GABA currents in

heterologous cells (Baulac et al. 2001; Ramakrishnan and Hess

2004), normal membrane traffic and synaptic aggregation were

observed in hippocampal neurons (Eugene et al. 2007).

Functionally, the K289M mutation accelerates the deactivation

of GABA currents by reducing mean channel open time (Bianchi

et al. 2002; Hales et al. 2006). In neurons, this effect accelerates

the decay of inhibitory synaptic currents (Eugene et al. 2007),

thereby reducing the efficacy of synaptic inhibition.

How may a general reduction of the efficacy of GABAergic

synapses specifically promote FS? In heterologous cells, an

increase in temperature has been shown to rapidly reduce

surface expression of K289M mutant c2. This reduction may

reflect increased receptor endocytosis and might contribute to

the emergence of FS in patients (Kang et al. 2006). However, in

the absence of synaptic specialization, the mechanisms in-

volved in this temperature-dependent endocytosis as well as its

relevance to synaptic GABA signaling are difficult to address in

heterologous cells. In neurons, postsynaptic receptor content

relies on 1) the insertion/internalization of receptors at the

membrane (Collingridge et al. 2004), 2) their lateral diffusion

into and out of synapses, and 3) their postsynaptic anchoring

through scaffold interactions (Triller and Choquet 2008).

GABAARs display free Brownian-type diffusion in extrasynaptic

membrane, confined movements within synapses, and rapid

translocation between these compartments (Jacob et al. 2005;

Thomas et al. 2005; Bogdanov et al. 2006; Levi et al. 2008;

Bannaı et al. 2009; Muir et al. 2010). In addition, the diffusion

properties of GABAARs are modulated by neuronal activity.

Receptor escape from synapses is facilitated by increased

excitatory synaptic activity, resulting in reduced synaptic re-

ceptor content and efficacy (Bannaı et al. 2009; Muir et al. 2010).

Since neuronal activity is strongly dependent on temperature

(e.g., Andersen and Moser 1995; Volgushev et al. 2000;

Postlethwaite et al. 2007), these observations predict increased

temperature may result in depletion of synaptic GABAARs.

Here, we report that raising temperature alters the post-

synaptic clustering of GABAARs and decreases GABA-mediated

miniature inhibitory postsynaptic current (mIPSC) frequency

in neurons expressing recombined K289M mutant but not

wild-type (WT) c2 subunit. This effect was associated with

a rapid increase in the lateral diffusion of synaptic K289M c2.This temperature-dependent increase in mutant c2 diffusion

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was mediated by enhanced excitatory transmission. These

results indicate that the K289M mutation in c2 confers synaptic

GABAARs with enhanced sensitivity to increased neuronal

activity that likely contributes to the involvement of this

mutation in FS.

Materials and Methods

Primary Hippocampal Cultures and TransfectionHippocampal neurons were prepared from E19 Sprague--Dawley rat

pups, as described (Eugene et al. 2007). Cells were plated at a density of

2.5 3 104 cells/cm2 and cultured in a CO2 incubator at 37 �C for 3--4

weeks in Neurobasal medium supplemented with B27 (Invitrogen,

Cergy Pontoise, France), 2 mM glutamine, and penicillin/streptomycin.

At 14 days in vitro, neurons were transfected with monomeric Red

Fluorescent Protein (mRFP) (0.118 lg/cm2) and recombinant Green

Fluorescent Protein (GFP)-tagged WT or K289M Gabrg2 constructs

(0.394 lg/cm2) using Lipofectamine (Invitrogen) according to manu-

facturer’s instructions (DNA:lipofectamine ratio 1:3 lg/lL) and used for

biological assays within 8--11 days posttransfection.

Temperature ExposureDue to the slow synaptic flux (exit/entry) of receptors, changes in

receptor lateral diffusion (5--10 min) precede changes in the density of

receptor at synapses (30--60 min) (Levi et al. 2008). Neurons were

therefore preincubated 10 min versus 1 h when studying the behavior

of molecules with single particle tracking (SPT) or that of receptor

population with cluster imaging and electrophysiology. For SPT

experiments, neurons were preincubated 10 min at 27, 31, 37, or 41

�C (depending on the experiment) in imaging medium (see below for

composition) following quantum dot (QD) labeling. They were then

used within 30 min. For cluster imaging, cells were preincubated in

culture medium 1 h in a CO2 incubator set at 41 �C. Cells were then

transferred to a recording chamber in imaging medium at the

appropriate temperature (37 or 41 �C) and used within 30 min (Figs

1 and 3E,F). For electrophysiology, neurons were preincubated in

culture medium 1 h in a CO2 incubator set at 27, 31, 37, or 41 �Cdepending on the experiment. Cells were then transferred to a re-

cording chamber in recording medium (see below for composition) at

31 �C (Figs 2 and 5) or at 27 �C (Figs 5 and 6). Neurons were then used

for experiments within 15 min, a time range too short to significantly

affect the number of postsynaptic receptors (Levi et al. 2008).

Live Cell Imaging and AnalysisCells were imaged in a temperature-controlled open chamber

(BadController V, Luigs & Neumann, Ratingen, Germany) mounted on

an inverted microscope (IX71 Olympus, Rungis, France) equipped with

a 360 objective (Numerical Aperture [NA] = 1.42, Olympus). GFP and

mRFP were detected using X-Cite 120PC lamp (EXFO, Mississauga,

Ontario, Canada) with appropriate filters (excitation: HQ470/40 and

D540/25, dichroic: Q495LP and 565DCLP, and emission: HQ525/50

and D605/55, Chroma Technology, Bellows Falls, VT). GFP and mRFP

images were acquired with an EMCCD camera (ImagEM; Hamamatsu

Photonics, Massy, France) using HC Image software (Hamamatsu).

Exposure time was determined on highly fluorescent cells to avoid

pixel saturation. All GFP and mRFP images from a given culture were

acquired with the same exposure time and acquisition parameters.

Quantification was performed using MetaMorph software (Roper

Scientific, Evry, France). A user-defined intensity threshold was applied

to select clusters and prevent coalescence. Data were obtained from 39

to 55 cells of 3 independent cultures.

Single Particle ImagingNeurons were incubated for 5 min at 37 �C with a rabbit primary

antibody against GFP (20--40 ng/mL, Roche Diagnostics, Meylan,

France), washed, and incubated for 5 min at 37 �C with a secondary

biotinylated Fab antibody (0.5 lg/mL, Jackson ImmunoResearch,

Newmarket, UK). Following washes, coverslips were then incubated

for 1 min at 37 �C with streptavidin-coated QDs emitting at 605 nm (0.5

nM, Invitrogen) in borate buffer (50 mM) supplemented with sucrose

(200 mM). Cells were then washed and imaged in the presence of

appropriate drugs after 5 min preincubation. All washes, incubation

steps, and cell imaging were performed in imaging medium prepared

with minimum essential medium without phenol red, supplemented

with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)

buffer (20 mM), glucose (33 mM), glutamine (2 mM), Na+ pyruvate

(1 mM), and B27 supplement (13) (all from Invitrogen).

Cells were imaged in a temperature-controlled open chamber

mounted on an Olympus IX71 inverted microscope equipped with

a 360 objective (NA = 1.42). GFP, mRFP, and QDs were detected using

X-Cite 120PC lamp with appropriate filters (excitation: HQ470/40,

D540/25, and D455/70; dichroic: Q495LP, 565DCLP, and 500DCXR;

and emission: HQ525/50, D605/55, and HQ605/20, GFP and mFRP

filters from Chroma Technology; QD filters from Omega Optical,

Brattleboro, VT). Real-time fluorescence images were obtained with an

integration time of 75 ms with the Hamamatsu ImagEM EMCCD camera

with 512 consecutive frames acquired under HC Image. Cells were

imaged within 30 min following primary antibody incubation. For each

SPT experiment, QDs dynamics were measured on 113 ± 13 QDs per

culture from 10 to 20 movies recorded from 2 separate coverslips per

culture. Data were obtained from 2 to 11 independent cultures. The

proportion of synaptic QDs was 25.9 ± 3.8% (average ± standard error

of the mean [SEM]) of the bulk population of QDs.

SPT and AnalysisSingle-molecule tracking was performed with custom software

(Bonneau et al. 2005) using Matlab (The Mathworks, Meudon, France).

Single QDs were identified by their blinking property (Alivisatos et al.

2005). The center of the fluorescence spots was determined with

a spatial accuracy of ~10 nm by cross-correlating the image with

a Gaussian fit of the point spread function (for details, see Triller and

Choquet 2008). QD trajectories were reconstructed as in Ehrens-

perger et al. (2007). Subtrajectories of single QDs with >20 points

without blinks were retained. Synaptic versus extrasynaptic trajecto-

ries were determined from overlay of trajectories image and GFP

image of GFP-coupled recombinant WT or K289M c2 clusters. GFP

images were first median-filtered (kernel size, 3 3 3 3 1) to enhance

cluster outlines. Then, a user-defined intensity threshold was applied

to select clusters and avoid their coalescence, and a binary mask was

generated. Trajectories were synaptic when overlapping with the

mask or extrasynaptic for spots 2 pixels (440 nm) away (Dahan et al.

2003). Diffusion coefficients were calculated from the longest

subtrajectories of single QDs in the synaptic and extrasynaptic

compartment. For each QD, we calculated the mean square

displacement (MSD) and diffusion coefficient (D) within extrasynap-

tic and synaptic compartments. The size of the confinement domain

and dwell time (DT) were calculated for synaptic QDs. Values of the

MSD plot versus time were calculated for each trajectory with the

following formula: MSD�ndt

�= 1N –n

+N –n

i=1

ððxi+n–xi�2+ðyi+n–yÞ2

�dt , where

xi and yi are the coordinates of an object on frame i, N is the total

number of frames in the trajectory, dt is the frame acquisition time,

and ndt is the time interval over which displacement is averaged

(Saxton 1997). For simple, 2D Brownian mobility, the MSD as

a function of time is linear with a slope of 4D, where D is the

diffusion constant. If the MSD as a function of time tends to a constant

value L, the diffusion is confined in a domain of size L. The diffusion

coefficient (D) is determined by a fit on the first 4 points of the MSD as

a function of time with MSD(ndt) = 4Dndt + b, where b is a constant

reflecting the spot localization accuracy. The area in which diffusion

is confined can be estimated by fitting the MSD as a function of time

with the following formula:MSD�ndt

�=L

2

3

�1–exp

�–12Dndt

L2

��+4Dmacndt ,

where L2 is the confined area in which diffusion is restricted and Dmac

is the diffusion coefficient on a longtime scale (Kusumi et al. 1993).

The size of the confinement domain was defined as the side of

a square in which diffusion is confined (Kusumi et al. 1993). For

details, see Ehrensperger et al. (2007). Synaptic DT was defined as the

duration of detection of QDs at synapses on a recording divided by the

number of exits as detailed previously (Charrier et al. 2006;

Ehrensperger et al. 2007).

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ElectrophysiologyNeurons were superfused with a recording medium containing (in

mM) 125 NaCl, 20 D-glucose, 10 HEPES, 4 MgCl2, 2 KCl, and 1 CaCl2(pH = 7.4) in a recording chamber maintained at 31 �C. mIPSCs were

recorded in whole-cell mode in the presence of TTX (1 lM), NBQX (20

lM), and D,L-APV (100 lM), with an internal solution containing (in

mM) 135 CsCl, 10 HEPES, 10 ethyleneglycol-bis(2-aminoethylether)-

N,N,N#,N#-tetra acetic acid (EGTA), 4 MgATP, 1.8 MgCl2, and

0.4 Na3GTP (pH = 7.4). Currents were recorded at –70 mV, with an

Axopatch 200B amplifier (Molecular Devices, Wokingham, UK), filtered

at 2 kHz and digitized at 20 kHz. Access and input resistance were

monitored with –5 mV voltage steps. mIPSCs were detected and

analyzed offline using Detectivent software (Ankri et al. 1994). In some

experiments, spontaneous synaptic (Fig. 5) or intrinsic (Fig. 6) activity

were recorded in voltage-clamp or current-clamp mode, respectively.

Spontaneous synaptic activity was recorded in the absence of any

postsynaptic receptor antagonist, with a solution containing (in mM)

115 CsMeSO4, 11.5 CsCl, 10 HEPES, 10 EGTA, 4 MgATP, 0.4 Na3GTP,

and 1.8 MgCl. Spontaneous intrinsic activity from a resting membrane

potential of –60 mV with a solution containing (in mM) 120 KMeSO4,

8 KCl, 10 HEPES, 10 EGTA, and 3 MgCl2.

Peptide Treatment and PharmacologyThe following peptides and drugs were used: myr-P4 (50 lM; Tocris

Bioscience, Bristol, UK), TTX (1 lM; Latoxan, Valence, France), NBQX

(10 lM), DL-AP5 (100 lM), R,S-MCPG (500 lM; Ascent Scientific,

Bristol, UK), and 4-AP (100 lM; Sigma-Aldrich, Lyon, France).

StatisticsData are presented as mean ± SEM. Means were compared using the

nonparametric Mann--Whitney rank-sum test unless otherwise stated.

Tests were performed using SigmaStat software (SPSS, Bois Colombes,

France). Cumulative distributions were compared using the Kolmo-

gorov--Smirnov test under StatView (SAS, Gregy-sur-Yerres, France).

Differences were considered significant for P values above 5%.

Results

Loss of Synaptic Aggregates of K289M Mutant c2 Subunitupon Temperature Elevation

Hippocampal neurons were transfected with either WT or

K289M (K289M) mutant GABAAR c2 subunit constructs with

GFP fused at their N-terminus. The postsynaptic aggregation of

recombinant c2 subunits was examined using live-cell imaging

of GFP in hippocampal neurons maintained at 37 �C. As

previously reported (Eugene et al. 2007), the K289M mutation

did not affect the membrane expression or aggregation of

recombinant c2. At 37 �C, numerous punctae of recombinant

c2 subunits were detected both on the soma and dendrites of

neurons expressing either WT or K289M c2 (Fig. 1A). Most

GFP-labeled recombinant c2 clusters were instantaneously

quenched by live exposure to bromophenol blue (5 mM),

indicating that recombinant c2 was inserted in the membrane

(Supplementary Fig. 1). Large recombinant c2 clusters were

mostly localized at inhibitory synapses, as revealed by their

close apposition to presynaptic varicosities immunoreactive for

the GABA synthesis enzyme, glutamic acid decarboxylase

(GAD; 73.7 ± 3.8%, n = 287, GAD positive synapses on

17 dendrites from 6 cells; data not shown).

In heterologous cells, several epilepsy-related mutations in

Gabrg2 have been suggested to result in reduced membrane

expression of c2 upon hyperthermia (Kang et al. 2006).

However, membrane trafficking and clustering of GABAAR are

likely differently regulated in heterologous cells and neurons.

We therefore compared the aggregation of recombinant c2 in

primary hippocampal neurons maintained for 1 h at 37 versus

41 �C. In these experiments, the survival of neurons was not

compromised as evidenced using the vital die trypan blue (not

shown). One-hour exposure to 41 �C had no detectable effect

on recombinant WT c2 clustering but induced a significant

decrease in the number of K289M mutant c2 clusters per 10

lm dendritic length (–27% of control, WT 37 �C, n = 2.2 ± 0.3

from 55 cells; WT 41 �C, n = 2.3 ± 0.3 from 50 cells; KM 37 �C,n = 2.2 ± 0.3 from 50 cells; KM 41 �C, n = 1.6 ± 0.3 from 39 cells;

3 cultures; P = 0.7 for WT and P < 0.05 for K289M c2; Fig. 1A,B).These observations suggest an increase in temperature of a few

degrees significantly reduce postsynaptic aggregation of

mutant but not WT c2 and predict a functional reduction in

the efficacy of synaptic inhibition in neurons expressing

K289M mutant c2.

Functional Impact of Temperature Rise on GABAergicSynaptic Transmission in Hippocampal Neurons

In order to examine the functional impact of the temperature-

induced reduction of clustering of mutant c2, we compared the

properties of mIPSCs in hippocampal neurons expressing

either WT or K289M mutant c2 (Fig. 2). mIPSCs were

pharmacologically isolated by tetrodotoxin (TTX, 1 lM) and

the glutamate receptor antagonists D,L-AP5 (100 lM) and

NBQX (20 lM). As previously reported (Eugene et al. 2007),

mIPSCs recorded from neurons expressing either WT

or K289M mutant c2 had similar frequency (11.4 ± 2.0 vs.

12.1 ± 1.0 Hz, P = 0.9), mean amplitude (–39.4 ± 5 vs. –34.8 ± 3.6

pA, P = 0.6), and onset kinetics (10--90% time to peak, 0.83

± 0.05 vs. 0.77 ± 0.04 ms, P = 0.33; n = 9--11 cells for both WT

and K289M) (Fig. 2A--C). However, their decay time constant

was accelerated by 28.8% (14.5 ± 1.0 vs. 10.3 ± 0.4 ms, P < 0.01)

in neurons expressing the K289M mutant as compared with

WT recombinant c2 (Fig. 2B,C). Therefore, in steady-state

conditions, the major synaptic effect of the K289M mutation

Figure 1. Loss of recombinant K289M but not WT c2 clusters upon raisingtemperature from 37 to 41 �C. ( A), Live-cell imaging of recombinant WT and K289Mc2 subunits in hippocampal neurons cotransfected with mRFP (24 days in vitro).Calibration, 5 lm. At 37 �C, large clusters of recombinant WT and K289M c2 can bedetected as GFP fluorescence spots. Note the loss of mutant but not WT c2 clustersafter 1-h exposure to 41 �C. mRFP images show dendrite outline of transfectedneurons. (B), The mean density of receptor clusters (number per 10 lm dendritelength) was reduced by 27% in neurons expressing K289M (black) as compared withWT c2 (white) after warming. Mann--Whitney rank-sum test, *P \ 0.05.

c2 K289M Mutation Alters GABAAR Diffusion d Bouthour et al.1544

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is to reduce current charge through GABAARs with no apparent

change in unitary conductance or mean number of receptor

per synapse (Eugene et al. 2007).

One-hour exposure to a temperature of 41 �C had no significant

effect on the mean amplitude, frequency, or decay kinetics of

mIPSCs in hippocampal neurons expressing recombinant WT

c2 (Fig. 2A--C). The mean amplitude of mIPSCs was also unaffected

by temperature rise in neurons expressing mutant K289M c2(36.2 ± 6.6 vs. 34.8 ± 3.6 pA) andwas comparable to that of neurons

expressing WT c2 (39.3 ± 4.5 vs. 39.4 ± 5.7 pA, P = 0.3; Fig. 2A--C),

suggesting the mean number of receptors per synapse was

unchanged. However, mIPSC frequency was reduced by 67% after

temperature increase in neurons expressing mutant K289M as

comparedwithWTc2(6.2±1.8vs. 18.6±5.1pA,P <0.05; Fig. 2A,C),suggesting that the number of inhibitory synapses containing

functional receptors was reduced. This is in agreement with

live-imaging data showing a reduced density of inhibitory synapses

with K289M c2 clusters upon temperature increase (Fig. 1).

If some synapses containing recombinant K289M c2 had lost

their postsynaptic receptors upon temperature elevation, then

the relative abundance of synapses containing only endogenous

WT receptors should increase. Since both types of receptors

can be distinguished based on their deactivation kinetics, we

would predict mIPSC decay would be slowed in neurons

expressing K289M mutant but not WT c2. Consistent with this

prediction, the mean decay time constant of mIPSCs recorded

in neurons expressing K289 mutant c2 increased after 1 h at

41 �C as compared with control (89 ± 5 vs. 71 ± 3% of WT,

P < 0.01; Fig. 2B,C). These results suggest that GABAARs

containing K289M mutant c2 subunit may escape from

inhibitory synapses upon temperature rise and/or may be

partially replaced by receptors containing endogenous WT c2.

Raising Temperature Promotes Synaptic Escape ofRecombinant GABAARs Containing K289M c2GABAARs diffuse laterally in the neuronal plasma membrane

and rapidly shift between extrasynaptic and synaptic sites

(Jacob et al. 2005; Thomas et al. 2005; Bogdanov et al. 2006;

Levi et al. 2008; Bannaı et al. 2009; Muir et al. 2010). Since

membrane dynamics properties control receptor content at

synapses (Choquet and Triller 2003; Triller and Choquet 2005,

2008), we asked whether enhancing temperature might

specifically affect the lateral diffusion of GABAARs containing

the K289M mutant c2 subunit.

The mobility of recombinant c2 was analyzed using QD-based

SPT (Dahan et al. 2003; Bannaı et al. 2006). The surface

recombinant c2 subunits were labeled with an antibody raised

against GFP and subsequently labeled with an intermediate

biotinylated Fab fragment and streptavidin-coated QD. We first

examined the impact of a rise in temperature on the lateral

diffusion of recombinant c2 subunits for bulk population of QDs

(i.e., independent of their synaptic vs. extrasynaptic localization).

Within 5--10 min after temperature reached 41 �C, the surface

explored by individual recombinant WT and K289M c2 was

reduced. Consequently, cumulative distributions of both WT and

K289M c2 diffusion coefficient (D) were shifted toward lower

values (WT 37 �C, D = 8.2 ± 0.5 3 10–2 lm2s

–1, n = 268; WT 41 �C,D = 6.7 ± 1.0 3 10

–2 lm2s–1, n = 270; KM 37 �C, D = 8.8 ± 1.8 3

10–2 lm2s

–1,n = 252; KM41 �C,D = 4.1 ± 0.33 10–2 lm2s

–1,n = 347;2 cultures; P < 0.001 for both WT and K289M c2; Fig. 3A).Protein traffic and endocytosis are temperature-dependent

processes and are accelerated at higher temperature. Receptors

undergoing endocytosis are immobilized in confined areas, such

as coated pits (Tardin et al. 2003; Petrini et al. 2009). Therefore,

the reducedmobility and increased confinement of bothWT and

K289M c2 upon raising temperature suggest that QD-stained

recombinant c2 may be trapped in endocytic membrane

domains. QD staining of receptors in SPT experiments pro-

vides access to only a small fraction of the entire membrane

population of receptors. This precludes visualization of newly

membrane-inserted receptors not yet engaged in internalization

and the lateral mobility of which may be sensitive to an increase

in temperature.

In order to circumvent this problem, we thus examined the

impact of hyperthermia on the lateral mobility of recombinant

c2 while pharmacologically blocking endocytosis using bath

application of myristoylated QVPSRPNRAP (myr-P4) peptide.

This peptide interferes with dynamin and amphiphysin in-

teraction (Marks and McMahon 1998), thereby blocking

Figure 2. Effects of raising temperature from 37 to 41 �C on synaptic GABA currentsin neurons expressing recombinant WT versus K289M c2. ( A), Continuous recordsand ( B), scaled averages of 100 GABAAR-mediated mIPSCs recorded fromhippocampal neurons transfected with WT (gray line) or K289M (black line) c2 incontrol conditions (37 �C) and after 1 h at 41 �C. Inset in B: Averaged mIPSC decayfitted by a single exponential (plain line). Calibration: 100 pA, 250 ms (continuousrecords); 20 ms (scaled averages); and 10 ms (averaged mIPSC decay fit). (C),Averaged mIPSC frequency, amplitude, and decay time constant from 9 to 11neurons transfected with recombinant WT (white) or K289M (black) c2 cultured at37 �C or after 1 h at 41 �C. mIPSC amplitude and frequency were not different at37 �C in neurons expressing recombinant WT or K289M c2. One-hour exposure to41 �C had no effect on the mean amplitude of mIPSCs in neurons expressing eitherWT or K289M mutant c2. However, mIPSC frequency was reduced by 67% inneurons expressing K289M mutant c2 as compared with WT c2. At 37 �C, mIPScsdecay faster in neurons expressing recombinant K289M versus WT c2. Thisdifference was significantly reduced after exposing neurons to 41 �C for 1 h. Mann--Whitney rank-sum test *P \ 0.05; **P \ 0.01. mIPSCs frequency, amplitude, anddecay time constant of neurons expressing recombinant WT and K289M c2 at 37 and41 �C were normalized to the corresponding averaged WT values measured at 37 �C.

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a crucial step for endocytosis (Wigge et al. 1997; Marsh and

McMahon 1999). In conditions of dynamin-dependent endocy-

tosis blockade, lateral diffusion of WT and K289M c2 did not

differ at 37 �C (WT 37 �C, D = 2.2 ± 0.2 3 10–2 lm2s

–1, n = 260,

KM 37 �C, D = 2.2 ± 0.4 3 10–2 lm2s

–1, n = 360; P = 0.03; 2

cultures; Supplementary Fig. 2). This is coherent with the

notion that, at steady states, the mutation does not change the

number of receptors per synapse (Eugene et al. 2007, Fig. 1).

Furthermore, myr-P4 prevented immobilization of QD-bound

c2 upon raising temperature to 41 �C (Fig. 3B), suggesting that

reduced diffusion observed at 41 �C in the absence of the

peptide reflected increased confinement of QDs within

clathrin-coated pits (Fig. 3A). However, in these conditions,

hyperthermia specifically accelerated diffusion of QD-bound

mutant (KM 41 �C, D = 3.8 ± 0.3 3 10–2 lm2s

–1, n = 403,

P < 0.001) but not WT c2 (WT 41 �C, D = 2.2 ± 0.3 3 10–2 lm2s

–1,

n = 339, P = 0.1; 2 cultures; Fig. 3B). Extrasynaptic versus

synaptic trajectories were segregated by comparison with GFP

images of c2 clusters (see Materials and Methods). Trajectories

were at inhibitory synapses when overlapping with c2 clusters

(e.g., red in Fig. 4A) or extrasynaptic (e.g., blue in Fig. 4A)

for trajectories 2 pixels (440 nm) away (Dahan et al. 2003).

We found that the increased mobility of K289M mutant c2at 41 �C was observed for both synaptic and extrasynaptic

receptors (synaptic: KM 37 �C, D = 2.1 ± 0.7 3 10–2 lm2s

–1,

n = 133; KM 41 �C, D = 3.8 ± 0.7 3 10–2 lm2s

–1, n = 114,

P < 0.001; extrasynaptic: KM 37 �C, D = 2.2 ± 0.2 3 10–2 lm2s

–1,

n = 192; KM 41 �C, D = 3.8 ± 0.6 3 10–2 lm2s

–1, n = 289,

P = 0.006; 2 cultures; Fig. 3C). Thus, lateral diffusion of GABAA

receptors containing mutant but not WT c2 subunit is

enhanced upon raising temperature from 37 to 41 �C.We then asked whether elevated temperature might relieve

constraints on K289M c2 diffusion at inhibitory synapses. The

MSD versus time relation for K289M c2 trajectories showed

a steeper slope, suggesting that trajectories were less confined

at inhibitory synapses at 41 versus 37 �C (data not shown).

Figure 3. Hyperthermia increases the lateral diffusion and decreases the postsynaptic clustering of K289M but not WT recombinant c2. ( A and B) Cumulative probabilities of QDdiffusion coefficients (for bulk population of QDs) associated with WT or K289M mutant c2 at 37 �C (gray) or 41 �C (black), in absence ( A) or presence (B) of the membrane-permeant dynamin inhibitory peptide P4 (myr-P4). In absence of myr-P4 (A) hyperthermia decreased diffusion coefficients of both recombinant WT and K289M c2. Kolmogorov--Smirnov test, ***P\ 0.001. In the presence of 50 lM myr-P4, (B) cumulative probability plots of diffusion coefficients of K289M but not WT c2 are shifted toward higher valuesat 41 �C. Kolmogorov--Smirnov test, ***P \ 0.001. (C) Diffusion coefficients of K289M are increased at 41 �C outside (left) and inside (right) inhibitory synapses. Kolmogorov--Smirnov test, ***P \ 0.001, **P \ 0.05. (D and E) Hyperthermia increases the size of the confinement domain (D) and decreases the synaptic DT (E) of K289M c2. Mann--Whitney rank-sum test, **P \ 0.005, ***P \ 0.001. (F and G) Live-cell imaging of recombinant WT and K289M c2 subunits in hippocampal neurons in presence of myr-P4.Calibration, 5 lm. At 37 �C, large clusters of recombinant WT and K289M c2 can be detected as GFP fluorescence spots (F). Note that the loss of mutant but not WT c2 clustersafter 1-h exposure to 41 �C. (G) The mean density of receptor clusters was reduced by 57% in neurons expressing K289M (black) as compared with WT c2 (white) afterwarming. Mann--Whitney rank-sum test, ***P \ 0.001.

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Accordingly, the mean size of the confinement domain (L) was

increased for K289M but not for WT c2 trajectories upon

raising temperature (WT 37 �C, L = 208.7 ± 18.1 nm, n = 84; KM

37 �C, L = 194.9 ± 17.3 nm, n = 125; WT 41 �C, L = 221.7 ± 37.3

nm, n = 38; KM 41 �C, L = 319.9 ± 41.0 nm, n = 91; 2 cultures;

P = 0.48 for WT and P = 0.002 for KM; Fig. 3D). Enhanced lateral

mobility and lower diffusion constraints on synaptic receptors

may alter the time receptors spend at synapses and thereby

influence synaptic receptor content (Triller and Choquet

2008). We asked whether temperature impact the time

GABAARs containing K289M mutant c2 spend at synapses.

A temperature jump from 37 to 41 �C did not alter DT of WT c2(WT 37 �C, DT = 18.6 ± 1.3 s, n = 154; WT 41 �C, DT = 18.8 ± 1.2

s, n = 189; 2 cultures; P = 0.89; Fig. 3E). In contrast, K289M c2DT were significantly decreased at 41 �C (KM 37 �C, DT = 21.3

± 1.3 s, n = 171; KM 41 �C, DT = 13.5 ± 1.5 s, n = 189; 2 cultures;

P < 0.0001; Fig. 3E), indicating a faster escape of mutant

receptors from the synaptic domain at 41 versus 37 �C.Reduced synaptic DT usually correlates with depletion of

postsynaptic receptor clusters (Bannaı et al. 2009; Charrier

Figure 4. Raising temperature from 27 to 31 �C increases the lateral diffusion of K289M but not WT recombinant c2 at inhibitory synapses. (A) Trajectories of GFP-coupled QDs(reconstructed from 38.4 s recording sequences) associated with recombinant WT or K289M c2 at 27 and 31 �C. Extrasynaptic trajectories are shown in blue and synapticportions in red. QD trajectories were overlaid with GFP fluorescence images of WT or K289M c2 clusters (green) in order to identify inhibitory synapses. Calibration, 1 lm. Notethat recombinant K289M but not WT c2 explored larger areas of inhibitory synapses at 31 �C versus 27 �C. (B, C, E, and F) Cumulative probabilities of QD diffusion coefficientsassociated with WT (B and C) or K289M mutant c2 (E and F) at 27 (dotted line) or 31 �C (plain line), outside (blue, B and E), or inside (red, C and F) inhibitory synapses. Insets inB and E: Cumulative probabilities of QD diffusion coefficients for bulk population of QDs. Note that the increase in diffusion coefficients of K289M but not WT c2 at 31 �C.Kolmogorov--Smirnov test, *P \ 0.05; ***P \ 0.001. (D and G) Impact of temperature increase from 27 �C (dotted line) to 31 �C (plain line) on averaged MSD as a function oftime for WT (D) and K289M (G) c2 at inhibitory synapses (red) and outside (blue). Raising temperature increased the slope of the MSD versus time function for K289M but notWT c2.

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et al. 2010). Since hyperthermia reduced postsynaptic aggre-

gation of mutant c2 (Fig. 1), we asked whether this reduction

was dependent on endocytosis of surface receptors. In the

presence of myr-P4 to block dynamin-dependent endocytosis,

1-h exposure to 41 �C significantly reduced (–57% of control)

the number of c2 clusters per 10 lm dendritic length in

neurons expressing the K289M mutant but not the WT subunit

(WT 37 �C, n = 2.5 ± 0.3 from 27 cells; WT 41 �C, n = 1.9 ± 0.2

from 30 cells; KM 37 �C, n = 2.4 ± 0.3 from 31 cells; KM 41 �C,n = 1.1 ± 0.2 from 27 cells; 2 cultures; P = 0.07 for WT and

P < 0.001 for K289M c2; Fig. 3F,G). We conclude that reduced

mutant c2 clustering observed at 41 �C did not result from

increased endocytosis but rather reflected a rapid escape of

receptors from synapses and receptor depletion from the

postsynaptic membrane.

Endocytosis is known to be a highly temperature-dependent

process. An alternative to pharmacological blockade of clathrin-

dependent endocytosis may then be to study c2 diffusion in

a lower temperature range. We thus examined the effects of

a rise in temperature from 27 to 31 �C on the diffusion of

recombinant c2 (Fig. 4). As observed at 41 versus 37 �C in the

presence of myr-P4, raising temperature from 27 to 31 �C did

not alter the exploratory behavior (Fig. 4A), diffusion coef-

ficients (synaptic: WT 27 �C, D = 2.7 ± 0.3 3 10–2 lm2s

–1, n = 83;

WT 31 �C, D = 3.0 ± 0.4 3 10–2 lm2s

–1, n = 81, P > 0.9;

extrasynaptic: WT 27� C, D = 4.6 ± 0.3 3 10–2 lm2s

–1, n = 362;

WT 31 �C, D = 4.7 ± 0.4 3 10–2 lm2s

–1, n = 246, P = 0.1; 11

cultures; Fig. 4B,C) or confinement (WT 27 �C, L = 339 ± 32 nm;

n = 39; WT 31 �C, L = 420 ± 45 nm; n = 38; from at least 3

cultures; P = 0.7; Fig. 4D) of WT QD-c2. In contrast, the

exploratory behavior (Fig. 4A) and the lateral mobility of the

mutant c2 subunit increased at 31 versus 27 �C for both

synaptic and extrasynaptic trajectories (synaptic: KM 27 �C,D = 2.7 ± 0.2 3 10

–2 lm2s–1, n = 177; KM 31 �C, D = 4.0 ± 0.3 3

10–2 lm2s

–1, n = 108; P < 0.001; extrasynaptic: KM 27 �C,D = 4.1 ± 0.2 3 10

–2 lm2s–1, n = 638; KM 31 �C, D = 4.7 ± 0.2 3

10–2 lm2s

–1, n = 479; 11 cultures, P = 0.03; Fig. 4E,F). The

steeper slope of the MSD plots for K289M c2 trajectories

(Fig. 4G) and the increase in the mean size of the confinement

domain L (KM 27 �C, L = 287 ± 18 nm, n = 84; KM 31 �C,L = 426 ± 32 nm, n = 50; >3 cultures; P = 0.01) illustrated

reduced confinement at inhibitory synapses at 31 versus

27� C. The time spent by K289M c2 at synapses was also

reduced after raising temperature from 27 to 31 �C (KM

27 �C, DT = 15.0 ± 1.0 s, n = 289; KM 31 �C, DT = 11.9 ± 1.0 s,

n = 217; >3 cultures; P = 0.09; data not shown). Therefore,

the membrane dynamics of K289M mutant but not WT c2subunit is sensitive to a rise in temperature both in the 27--31

and 37--41 �C range. We therefore used this lower temperature

range in experiments to further elucidate the mechanisms

involved in this phenomenon.

Increased Lateral Diffusion of K289M c2 upon WarmingInvolves Excitatory Synaptic Activity

Most biological processes are temperature dependent. In

particular, increased temperature may affect the fluidity of

the plasma membrane and thereby influence lateral diffusion

of transmembrane proteins, such as GABAARs. Alternatively,

temperature may act to increase synaptic transmission (Schiff

and Somjen 1985; Moser et al. 1993; Volgushev et al. 2000) and

indirectly promote activity-dependent modulation of postsynaptic

receptor diffusion (Levi et al. 2008; Bannaı et al. 2009; Muir et al.

2010). We asked whether the effect of raising temperature on

the lateral diffusion of c2 may be intrinsic or rather mediated

by postsynaptic activity.

We first examined the effect of raising temperature in the

range 27--31 �C on spontaneous synaptic activity in hippocam-

pal neurons. Mixed excitatory and inhibitory spontaneous

postsynaptic currents (spPSCs) were recorded in hippocampal

neurons first at 27 �C and 15--20 min after raising temperature

to 31 �C (Fig. 5A,B). SpPSC frequency increased from 20% to

146% in 5 of 6 recorded neurons (mean frequency = 161.6 ±22.4% of control, n = 6, Wilcoxon signed-rank test P < 0.05).

Therefore, even a modest change in temperature was sufficient

to induce a rapid increase in spontaneous synaptic activity. This

is in agreement with previous data showing enhanced synaptic

excitatory transmission after raising the brain temperature

from 29 to 33 �C (Schiff and Somjen 1985).

Since GABAAR membrane dynamics are regulated by synaptic

activity (Levi et al. 2008; Bannaı et al. 2009; Muir et al. 2010),

we asked whether blocking intrinsic or synaptic activity may

prevent the increased diffusion of K289M mutant c2 upon

raising temperature. We compared the effect of raising

temperature from 27 to 31 �C in the presence or absence of

the sodium channel blocker TTX alone (1 lM), D,L AP5 (100

lM) alone or in combination with TTX (1 lM) and other

glutamate receptor antagonists (NBQX, 10 lM; D,L-AP5, 100

lM; and R,S-MCPG, 500 lM; Fig. 5C--E). TTX alone did not

prevent the temperature-induced acceleration of K289M

mutant c2 (Control, KM 27 �C, D = 6.9 ± 0.8 3 10–2 lm2s

–1,

n = 273; Control, KM 31 �C, D = 7.4 ± 0.4 3 10–2 lm2s

–1, n = 345;

TTX, KM 31 �C, D = 8.0 ± 0.4 3 10–2 lm2s

–1, n = 247; 3 cultures;

P < 0.001 for KM 27 vs. 31 �C and P = 0.01 for KM 31 �C vs.

KM31 + TTX; Fig. 5C--E). In contrast, application of D,L AP5

alone completely abolished the effect of temperature on the

diffusive properties of K289M mutant c2 (D,L-AP5, KM 31 �C, D= 4.2 ± 0.3 3 10

–2 lm2s–1, n = 345; P < 0.001; Fig. 5C,E).

Application of all antagonists reduced the diffusion of the

recombinant subunit below that observed in control conditions

(TTX + AP5 + NBQX + MCPG, KM 31 �C, D = 3.2 ± 0.3 3 10–2

lm2s–1, n = 212; 3 cultures; P < 0.001; Fig. 5C,E). These results

suggest that the K289M mutation potentiates the sensitivity of

lateral diffusion of the receptor to excitatory synaptic activity.

This conclusion predicts the K289M mutation in c2 may

increase the lateral diffusion of GABAARs when excitatory

synaptic activity is enhanced, independent of a rise in

temperature. We examined this issue by comparing the

membrane dynamics of recombinant WT and K289M c2subunits before and after application of the potassium channel

blocker 4-aminopyridine (4AP, 100 lM). In untransfected

neurons recorded at 27 �C, application of 4AP rapidly led to

a robust increase in both spPSP frequency and firing that

persisted throughout the application of the drug (Fig. 6A).

Although 4AP has previously been shown to increase lateral

diffusion of endogenous GABAARs in hippocampal neurons

(Bannaı et al. 2009 and Supplementary Fig. 3), it had little or no

effect on the confinement (Fig. 6B) or diffusion coefficients of

recombinant WT c2 (Control, WT 27 �C, D = 4.4 ± 0.3 3 10–2

lm2s–1, n = 398; 4AP, WT 27 �C, D = 4.6 ± 0.5 3 10

–2 lm2s–1, n =

201; 2 cultures; P = 0.75; Fig. 6B,C). In contrast, 4AP

significantly increased the exploratory behavior (Fig. 6B) and

diffusion coefficient of K289M mutant c2 (Control, KM 27 �C,D = 3.0 ± 0.2 3 10

–2 lm2s–1, n = 509; 4AP, KM 27 �C, D = 4.1 ±

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Figure 5. Blocking excitatory synaptic transmission reverses the effect of raising temperature on K289M c2 lateral diffusion. ( A) Continuous records of spPSCs from hippocampalneurons maintained at 27 or 31 �C. Calibration, 200 pA, 200 ms. (B) spPSC frequency increased by ~60% upon raising temperature from 27 to 31 �C. (C) Examples of trajectoriesof recombinant QD-coupled K289M c2 at 27 �C (361 frames), at 31 �C in absence of drugs (484 frames) or in the presence of TTX, AP5, NBQX, and MCPG (285 frames) or D,LAP5 alone (376 frames). Calibration, 1 lm. (D and E) The increase in diffusion coefficients of the bulk population of K289M c2 following temperature increase from 27 �C (plaingray) to 31 �C (black plain) was reversed by addition of D,L AP5 alone (doted black line) and further prevented by addition of TTX, AP5, NBQX, and MCPG (dashed black line) butnot by addition of TTX alone (dashed and doted black line). Kolmogorov--Smirnov test, ***P \ 0.001.

Figure 6. The potassium channel blocker 4-aminopyridine mimics the effect of raising temperature on the lateral diffusion of K289M mutant c2. ( A) Continuous current-clamprecording of a 22 days in vitro neuron during application of 100 lM 4AP. Right enlarged recording sections showing large spPSCs and cell discharge in the presence of 4AP.Calibration, 20 mV, 2 min (continuous record); 20 mV, 1 s (insets). (B) Trajectories of recombinant c2 at 27 �C in absence (WT, 146 frames; K289M, 378 frames) or in presenceof 100 lM 4AP (WT, 419 frames; K289M, 257 frames). Calibration, 1 lm. Note that the increase in the explored area of mutant but not WT c2 in the presence of 4AP. (C)Cumulative probabilities of diffusion coefficients of the bulk population of WT (left) or K289M (right) c2 at 27 �C in the absence (gray) or presence (black) of 4AP. Note theincrease in diffusion coefficients of K289M but not WT c2 in the presence of 4AP. Kolmogorov--Smirnov test, ***P \ 0.001.

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0.3 3 10–2 lm2s

–1, n = 330; 2 cultures; P < 0.001). Therefore,

GABAARs containing the recombinant K289M mutant but not

WT c2 subunit show enhanced lateral diffusion during

sustained neuronal activity. Altogether, these results demon-

strate that the K289M mutation in the c2 subunit confers

synaptic GABAARs with enhanced sensitivity to increased

neuronal activity. We conclude GABAA receptors containing

K289M mutant c2 show increased diffusion and faster escape

from synapses in conditions of enhanced neuronal activity,

such as upon temperature elevation.

Discussion

We have shown that the K289M mutation in the c2 subunit

affects the membrane dynamics and postsynaptic aggregation

of GABAARs in conditions of increased temperature or neuronal

activity. Raising temperature reduced the clustering of mutant

c2 and decreased the efficacy of synaptic inhibition in neurons

expressing mutant but not WT recombinant c2. A rise in

temperature, in conditions of reduced endocytosis, increased

the diffusion coefficients and decreased the confinement and

synaptic DT of K289M c2, thereby favoring its escape from

synapses. This effect was likely due to the enhanced neuronal

activity induced by the temperature rise since the increase in

K289M c2 dynamics was reversed by pharmacological blockade

of excitatory synaptic transmission and mimicked by the

convulsant drug 4-aminopyridine. We conclude the K289M

mutation in c2 confers GABAARs with enhanced sensitivity to

neuronal activity that may then trigger the escape of receptors

from synapses and thereby further reduce the efficacy of

GABAergic inhibition. We suggest that this reflects a conforma-

tional change of c2 that may impair receptor--scaffold

interactions at synapses.

Temperature-Induced Loss of Mutant K289M c2 Clustersat Inhibitory Synapses

We have shown that postsynaptic receptor clusters containing

the K289M mutant c2 rapidly disappear upon temperature

increase. This effect was detected as a reduced number of GFP

punctae per 10 lm dendritic length in neurons expressing

recombinant GFP-Gabrg2 bearing the K289M mutation. It was

correlated with a reduced frequency of mIPSCs suggesting the

number of functional GABAergic synapses was reduced. This

effect is unlikely to reflect changes in presynaptic function

since it was only observed in neurons expressing recombinant

mutant but not WT c2. Currents carried by GABAARs

containing K289M mutant c2 show faster decay (Eugene

et al. 2007), reflecting faster deactivation kinetics (Bianchi

et al. 2002; Hales et al. 2006). We took advantage of this

physiological signature to compare the relative proportion

of synapses containing the mutant subunit before and after

heating. Whereas mIPSC decay was significantly faster in neu-

rons expressing K289M mutant versus WT c2 at 37 �C, thisdifference became nonsignificant after 1-h exposure to 41 �C,suggesting the contribution of synapses devoid of mutant c2 to

mIPSCs had increased.

This observation reveals that all GABAergic synapses do not

express receptors containing recombinant c2 in transfected

neurons. Consistent with this conclusion, a fraction of GAD

immunopositive terminals were not facing GFP clusters in

neurons expressing recombinant c2 and yet colocalized with

endogenous c2 clusters (data not shown). The specificity of

recombinant c2 synaptic incorporation may reflect distinct

behaviors of the 2 splice variants c2S and c2L that are

expressed in hippocampal neurons (Gutierrez et al. 1994;

Khan et al. 1994; Miralles et al. 1994). The present study was

conducted using recombinant c2L. This variant might be

preferentially associated with synaptic GABAARs (Baer et al.

2000) and differs from c2S by an extra 8 amino acids within the

cytoplasmic loop with putative phosphorylation site by protein

kinase C (Moss et al. 1992). Postsynaptic aggregation of c2requires the anchoring protein gephyrin (Essrich et al. 1998;

Kneussel et al. 1999, 2001; Levi et al. 2004) and c2L- but notc2S-gephyrin interaction, and postsynaptic clustering is regu-

lated by Protein Kinase C (Meier and Grantyn 2004). Therefore,

c2S and c2L clustering to distinct postsynaptic sites may reflect

different interactions with gephyrin. Alternatively, c2 isoforms

may be part of heteropentamers with different content in asubunits with various binding properties to scaffolding

molecules (Tretter et al. 2008).

Although mIPSC frequency was reduced upon raising

temperature, likely reflecting a loss of postsynaptic receptors

at synapses containing mutant c2, the mean amplitude of

mIPSCs was unaffected. This may be explained if 1) the affinity

of scaffolding molecules for GABAARs somehow acts to

maintain receptor content constant by replacing mutant c2containing receptors by receptors containing endogenous c2or 2) most synapses containing mutant c2 were totally depleted

from postsynaptic receptors after 1 h at 41 �C. Both hypotheses

predict that mIPSC decay time would increase after heating in

neurons expressing mutant c2 (Fig. 2). However, it seems

unlikely that inhibitory synapses maintained their postsynaptic

receptor content since a partial reduction in synaptic GABAAR

content can be achieved in conditions of increased neuronal

activity (Bannaı et al. 2009; Muir et al. 2010). We conclude that

exposure of neurons to 41 �C very rapidly leads to a complete

loss of postsynaptic GABAARs containing K289M mutant c2 at

some, but not all, inhibitory synapses.

Facilitation of Mutant c2 Diffusion at Inhibitory Synapsesby Neuronal Activity

We report that raising temperature by a few degrees is

sufficient to increase lateral diffusion of mutant but not WT

recombinant c2 in hippocampal neurons. Accelerated mem-

brane turnover including endocytosis of individual GABAARs

precluded single-molecule tracking experiments to be con-

ducted at 41 �C. Indeed, both WT and KM recombinant c2 were

slowed down and confined upon hyperthermia. This reduced

diffusion likely reflected increased trapping of receptors in

endocytotic pits since pharmacological blockade of dynamin-

dependent endocytosis revealed increased diffusion and de-

creased synaptic DT of K289M recombinant c2. Reduction in

synaptic DT often correlates with depletion of postsynaptic

receptor clusters (Bannaı et al. 2009; Charrier et al. 2010). The

loss in postsynaptic receptor content is likely due to reduced

receptor trapping by the subsynaptic scaffold. This may reflect

a reduction in 1) the number of gephyrin molecules at synapses

(Bannaı et al. 2009; Charrier et al. 2010), 2) the receptor ability

to interact with gephyrin (Zita et al. 2007; Levi et al. 2008),

and/or 3) gephyrin oligomerization (Bedet et al. 2006; Calamai

et al. 2009; Charrier et al. 2010). Temperature-induced loss of

K289M c2 clusters was also detected after blockade of

dynamin-dependent endocytosis, suggesting that reduced

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clustering of mutant c2 at 41 �C did not result from increased

internalization but rather reflected a rapid escape of receptors

from synapses and receptor depletion from the postsynaptic

membrane.

How might temperature influence lateral diffusion and

clustering of synaptic GABAARs? Most biological processes

may be accelerated at higher temperatures yet neuronal

activity seemed most likely to mediate this effect. While action

potentials may be reduced in amplitude and/or frequency at

higher temperatures (Andersen and Moser 1995; Volgushev

et al. 2000), synaptic transmission on the contrary is enhanced

(Katz and Miledi 1965; Schiff and Somjen 1985; Moser et al.

1993). This latter effect involves a combination of presynaptic

(increased vesicle pool recycling, Pyott and Rosenmund 2002;

Kushmerick et al. 2006) as well as postsynaptic factors

(acceleration of postsynaptic receptor activation, Postlethwaite

et al. 2007). Consistent with these observations, we observed

an increased frequency of spPSCs in hippocampal neurons

upon raising temperature. Increased lateral diffusion of mutant

c2 at higher temperatures was reversed by the NMDA receptor

antagonist D,L-AP5 and even more so by a combination of

glutamate receptor antagonists and TTX. On the other hand,

TTX alone had little or no effect, while 4-aminopyridine

mimicked the effect of temperature. These results point to

a major role of postsynaptic glutamate receptor activation on

the temperature-dependent modulation of mutant c2 diffusion.

Accordingly, GABAAR lateral diffusion has previously been shown

to be strongly dependent on postsynaptic NMDAR activation

and Ca2+-calcineurin signaling (Bannaı et al. 2009; Muir et al.

2010). In particular, diffusion coefficients of synaptic GABAARs

were directly correlated with intracellular Ca2+concentrations.

This modulation might involve a Ca2+-dependent modulation

of either the stability of gephyrin scaffold (Hanus et al. 2006)

or the receptor--gephyrin interactions through the a2 sub-

unit (Tretter et al. 2008), or both. Noticeably, this modulation

seems to differentially affect receptors containing endogenous

versus recombinant WT c2 (Fig. 6 and Supplementary Fig. 3).

The greater sensitivity to excitatory synaptic activity of endo-

genous as compared with recombinant WT c2 diffusion will

need to be further explored and might reflect differences

between splice variants.

We show that the sensitivity of GABAARs to Ca2+-dependent

modulation of their diffusion is enhanced by the K289M

mutation in c2. This effect might reflect an indirect increase in

neuronal excitation due to the reduced efficacy of synaptic

inhibition in neurons expressing the mutant c2 (Eugene et al.

2007; Fig. 2). However, in this case, one would expect faster

diffusion and reduced clustering of K289M versus WT c2 even

at steady state. Instead, the K289M mutation alters neither

receptor clustering nor inhibitory synaptic transmission at

37 �C (Eugene et al. 2007; Figs 1 and 2). This argues against

an indirect impact of the mutation on c2 diffusion through

enhanced excitatory drive. Instead, we propose the mutation

per se may act to affect the allosteric conformation of the

receptor. The K289 residue is lining the external mouth of the

pore of the channel, between transmembrane segments II and

III (O’Mara et al. 2005). Therefore, it seems unlikely the K to M

mutation may directly compromise gephyrin--receptor or c2-a2interactions. However, the mutation is known to affect

receptor allosteric conformation and favors its closed state

(Bianchi et al. 2002; Hales et al. 2006). We thus propose this

allosteric conformation may unmask a region or residue of c2

that controls its diffusion (e.g., Muir et al. 2010). Therefore,

favoring the closed state of GABAARs would enhance the

sensitivity of their lateral diffusion to Ca2+and promote their

escape from inhibitory synapses.

Gabrg2 Mutations and the Mechanisms of Febrile Seizures

Our results demonstrate the K289M mutation in the GABAAR

c2 subunit affects GABAergic signaling in 2 distinct ways

depending on the level of neuronal activity. We had previously

shown expressing K289M recombinant c2 in hippocampal

neurons results in accelerated IPSC kinetics, which likely leads

to both smaller and faster IPSPs in these cells (Poncer et al.

1996; Eugene et al. 2007). This effect is dominant since it was

observed in neurons expressing endogenous WT c2. In

addition, we now show the K289M mutation also increases

the sensitivity of lateral diffusion of synaptic GABAARs to

neuronal activity. This effect does not result in a significant

difference in membrane expression or clustering of mutant c2as compared with WT (Eugene et al. 2007 and this study). This

suggests that the level of spontaneous activity in primary

hippocampal cultures may not be sufficient to lead to a steady-

state decrease in synaptic clustering of K289M mutant c2.Instead, this effect may become prominent only in conditions

of increased neuronal activity, sufficient to induce activation of

postsynaptic NMDARs. This may occur during febrile episodes,

which lead to respiratory alkalosis and subsequent neuronal

hyperexcitability (Schuchmann et al. 2006). It may then further

attenuate synaptic inhibition by reducing the number rather

than the efficacy of functional GABAergic synapses and thereby

precipitate seizures.

The mechanisms by which fever leads to seizures remain

poorly understood. Enhanced temperature-dependent endocy-

tosis has been proposed as a common mechanism for all

Gabrg2 mutations associated with FS in humans (Kang et al.

2006). Although attractive, this hypothesis seems unlikely to

account for the involvement of other Gabrg2 mutations in FS.

In neurons, the R43Q mutation largely compromises mem-

brane targeting of c2 and prevents synaptic aggregation with

other subunits (Eugene et al. 2007; Frugier et al. 2007; Tan et al.

2007). Instead, this mutation mostly reduces GABA signaling

through a reduction of tonic inhibition. Similarly, although

some surface expression of the Q351X mutant c2 was reported

in heterologous cells, none is detected in neurons expressing

this mutant (Eugene et al. 2007). Therefore, the possible

contribution of an activity-dependent escape from synapses of

these mutant c2, similar to that described here for the K289M

mutant, seems unlikely to account for the functional pheno-

type associated with these mutations. Other c2 mutations

associated with FS (R139G, Audenaert et al. 2006; W390X, Sun

et al. 2008) will need to be further explored in neurons to

confirm whether the mechanism described in the present

study can be generalized to other mutants.

Supplementary Material

Supplementary material can be found at: http://www.cercor

.oxfordjournals.org/

Funding

Avenir program of Institut National de la Sante et de la

Recherche Medicale (to J.C.P.) and grants from the city of Paris;

Cerebral Cortex July 2012, V 22 N 7 1551

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the Fondation Electricite de France; and the Fondation pour la

Recherche Medicale (to J.C.P).

Notes

We thank Steve J. Moss and Christel Depienne for kindly providing the

original WT and K289M mutant Gabrg2-GFP constructs and Norbert

Ankri for sharing and assistance with Detectivent software. Conflict of

Interest : None declared.

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